豬隻體內兒茶素之抗氧化效能與腸道作用研究

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1 豬隻體內兒茶素之抗氧化效能與腸道作用研究
中文摘要 茶是人類長期飲用的飲料，其中兒茶素(Catechins)佔茶粉抽提成分中20％以上，為主要的生理活性成分，主要成分為(-)-epigallocatechin gallate (EGCG)。兒茶素抗氧化之能力被認為是其抗細胞突變、防癌、預防心血管疾病的主因，但口服兒茶素之絕對生體可用率不到10%，因此未被吸收之兒茶素對腸道之效能亦應有所了解。基於預防醫學的觀點，體內兒茶素的抗氧化作用與其對腸道菌相之影響，是要長期觀察探討。本研究藉由長期餵予豬隻即溶綠茶粉，觀察豬隻體內血中自由基生物指標物質（MDA）與體內抗氧化生物指標物質（GSH）的增減，並檢測兒茶素的血中濃度相互評比，以明確瞭解兒茶素其抗氧化之功能。再藉由分析兒茶素成分，確認兒茶素之藥物動力學與其生體可用率，合併觀察未被吸收之兒茶素對腸內菌相的影響。 在兒茶素之抗氧化效能方面：豬隻連續口服Catechins即溶綠茶粉前後，在血漿中MDA濃度並無明顯增減趨勢，然而，血中GSH濃度則有明顯增加之趨勢。將口服投予即溶綠茶粉之後十九天內，生物指標物質的濃度與對照組（即投藥前三天之平均值）比較，發現血漿中MDA與GSH濃度並無明顯增減趨勢，且統計上無顯著差異存在；但於全血內GSH的濃度則有明顯增加之趨勢( P=0.013 )。由此得知在每日高劑量即溶綠茶粉口服投予下，未能影響血中MDA濃度，但血中GSH濃度有增加之趨勢，尤其以全血中還原型GSH濃度有較明顯的上升。 一簡單、精確且具有專一性的HPLC分析方法，已被發展用以同時偵測生物檢品中兒茶素成分（EGCG與ECG）的濃度。利用逆相層析管，配合280 nm之紫外光波長偵測，運用單一流動相合併使用不同的流速進行偵測。EGCG在血漿濃度5至200mg/ml的範圍內呈現良好線性關係（Y= X，r2=0.9920），ECG在血漿濃度10至200mg/ml的範圍內呈現良好線性關係（Y= X，r2=0.9970），同日內與異日內分析之CV值均在可接受範圍，顯示此為良好之分析方法。在8.64mg/kg劑量之靜脈投予六隻豬，分析其血中濃度，顯示EGCG與ECG符合二室性模式之藥物動力學性質。其排除半衰期分別為226.0±161.5、315.1±504.0分鐘，分布體積分別為34.4±21.0、43.8±65.0 ml/kg，清除率分別為0.11±0.35、0.10±0.40 ml/min/kg，而曲線下之面積（AUC）分別為12.0±3.4、17.0±10.1 mg×min/ml。 兒茶素對腸道之影響：豬隻連續口服Catechins即溶綠茶粉前後，腸道之總菌落數 (3.2±1.8 vs. 3.6±2.3)、厭氧性菌總菌落數 (3.8±1.9 vs. 3.5±1.8) 並無明顯之變化，有害菌大腸桿菌數 (6.3±3.1 vs. 4.8±1.9) 未見增減；然而，益生菌雙叉桿菌數 (1.6±1.2 vs. 6.7±3.0) 在投予即溶綠茶粉第八天後，具有顯著增加趨勢，其菌落數約為投予前之2-10倍。投予即溶綠茶粉第十二天後，乳酸桿菌菌落數 (4.2±2.4 vs. 8.9±1.9)顯著的微增，為未投予時之2倍。結果顯示益生菌雙叉桿菌及乳酸桿菌在腸菌叢中比例增高時，即相對的縮減有害菌的生存空間；雖然豬樣本少且腸道菌的差異性大，結果之標準偏差值較大，卻具有統計上顯著差異，因此口服連續投與高劑量即溶綠茶粉確實能改善腸道菌相，有利於腸道的正常生理機能。 本試驗連續口服即溶綠茶粉於豬隻體內，同時觀察其體內氧化壓力與腸道菌相之變化，發現長期服用即溶綠茶粉雖未能明顯降低體內脂質過氧化物（MDA）的產生，但卻能有效增加抗氧化物質（GSH）之含量；而且未被吸收之綠茶也能在腸道中明顯提高益生菌種（雙叉桿菌與乳酸桿菌）之數目，在有限的腸道空間內有害菌的生長即被抑制。因此口服連續投與高劑量兒茶素不僅能顯著提昇生理之抗氧化作用，也確實能改善腸道菌相有利於腸道的正常生理機能。

2 The influence of catechins on the antioxidative activities and enterobateria in pigs.
英文摘要 More than 20% of catechins are in the extracted tea powder and (-)-epogallocatechin gallate (EGCG) is the major component of catechins. The antioxidative activity is thought as the main force to their antimutagenic, anticancer and protection of cardiovascular disease. While, The oral absolute bioavailability of catechins was less than 10%. Therefore, understanding the influence of unabsorbed catechins in GI tract became necessary. In the protecting medicine point, it is necessary to evaluate that the influence the antioxidative activity of catechins and catechins on the enterobacteria in pigs of a long term. The aim of this study is to evaluate the antioxidant activities of catechins and the influence of catechins on the enterobacteria in pigs. Evaluation the antioxidative activity of catechins：After oral catechins 19 days, the plasma concentration of MDA did not changed while GSH concentration have an increasing tendency. Comparison of before and after oral catechins with t-test, plasma MDA concentration and plasma GSH concentration didn’t have statistical significant different. However, the GSH concentration in whole blood has statistical significant different（p=0.013）. Therefore, we infer that daily oral administration of high dose of catechins would increase the GSH concentration and enhance the ability of defense system. daily administration of high dose (520mg/kg, containing EGCG 60 mg/kg), but it did not change the MDA concentration, it infers that the damage of free radical causing from lipid peroxidation did not significant decreased or increased. A simple, accuracy and specific analytical method based on HPLC was developed to detect the catechins in the biological sample. A C8 reversed phase column with UV detection at 280nm was used in chromatographic separation. Under the chromatographic conditions, the calibration curve of EGCG and ECG in plasma shows a good linearity within the concentration range of 5 to 200mg/ml（Y= , r2=0.9920）and 10 to 200mg/ml（Y= , r2=0.9970）. the validation of this analytical method are within the acceptable criteria. After intravenous administration（I.V.）of 8.64mg/kg green tea, the plasma concentration-time profiles of EGCG and ECG would be described by a bi-exponential equation. the elimination half-life are 226.0±161.5、315.1±503.9min, the distribution volume are 34.4±21.0、43.8±65.0 ml/kg，the clearance are 0.11±0.35、0.10±0.40 ml/min/kg，the area under the curves（AUC）are 12.0±3.4、17.0±10.1 mg×min/ml。 The influence of catechins on the enterobacteria：comparing with the colony before and after administration of catechins, total aerobic bacteria (3.2±1.8 to 3.6±2.3) and anaerobic bacteria (3.8±1.9 to 3.5±1.8) and E. coli (6.3±3.1 to 4.8±1.9) was not significantly changed. However, Bifidobacterium spp. and Lactobacillius spp. have an increasing tendency beginning at eighth day. Bifidobacterium spp. increased significantly and reached to 2 to 10 times of baseline data (1.6±1.2 to 6.7±3.0). Lactobacillius spp. increased slightly two times of control from the twelfth day (4.2±2.4 to 8.9±3.2). The ratio of colony of Bifidobacterium spp. to E. coli was increased accompanying to the times of administration. It indicated that catechins would be helpful to reconstruct or improve the normal enterobacteria in pigs by increase the Bifidobacterium spp.. In this study, after catechins administration daily, the blood GSH was also significantly increased and Bifidobacterium spp. and Lactobacillius spp. have an increasing tendency. Therefore, we inferred that oral administration of high dose of tea catechins daily would enhance the ability of defense system by increased whole blood GSH concentration and improve the GI flora by increase the Bifidobacterium spp. and Lactobacillius spp. to have the benefit to bowel normal conditions.